KR19980086562A - Glass microfiber column and plasmid DNA production method using the same - Google Patents

Abstract

The present invention relates to a glass microfiber column and a method for preparing plasmid DNA using the same, and by constituting the column with borosilicate glass microfiber, a large amount of high-purity plasmid DNA can be obtained simply and quickly without additional purification process, and also according to the present invention. The kit can be used to effectively separate DNA from agarose gels or polyacrylamide gels, making it an excellent invention for biotechnology research.

Description

Glass fine fiber column and manufacturing method of plasmid DNA using the same

The present invention relates to a column kit for preparing plasmid DNA. More specifically, the present invention relates to a method for producing a column kit, which can be obtained from borosilicate or silicate glass microfibers in a large amount of high purity plasmid DNA in a short time, and to a method for producing plasmid DNA using the column kit.

The method of preparing plasmid DNA is the most widely required technique in molecular biology research. Conventionally, methods for preparing plasmid DNA are known. In many laboratories, plasmids are manufactured according to the manual method, which is time-consuming and unreliable, and in laboratories with good research conditions, products made in Qiagen (Germany) or Promega (US) are mainly used. These conventional products mainly use a method in which plasmid DNA is bound to silica gel. On the other hand, according to Vogelstein and Gillespie's paper (1978), flint glass is formed by particle size of large flint glass particle, medium flint glass particle, and flint glass powder, and then bound to flint glass by measuring DNA bound under NaI solution. Methods of separating DNA from agarose gels are known. The product based on this principle is already used as a product called 'glass milk' and is useful for separating DNA from agarose gel. However, the effect of DNA separation according to this product has the most effective DNA binding ability in flint glass powder, but the effect is sharply degraded in medium flint glass particles, and the effect is more remarkably reduced in large flint glass particles. In addition, the large borosilicate glass particles of different glass materials, as in the case of large flint glass particles, DNA binding ability is not effective, the glass fiber filter has a very low adsorption force, so the yield in a small volume is very low. And when using porous glass bead showed excellent DNA binding activity, but it takes a long time to bind DNA, there is a degradation of DNA when obtained from the bead, there is a problem that the yield is also not suitable for quantitative analysis. The silica gel shows a very effective binding effect of DNA as in flint glass powder, but has a problem of handle mechanically and a low yield. Silica gel is used as a column kit for the production of plasmid DNA, and 'Qiagen' and 'Promega' products are commercially available. However, these products are cumbersome and require extra purification process in the plasmid DNA manufacturing process. Also, importing these foreign products requires a lot of cost. Therefore, low-cost plasmid DNA can obtain a large amount of high purity plasmid DNA by simple process. There is an urgent need for production column kit development.

Therefore, an object of the present invention is to obtain a large amount of high-purity plasmid DNA in a short time without a separate purification process, plasmid DNA production column that can be widely used for DNA digestion, ligation, cloning, CAT assay, 35S sequencing, automatic sequencing, cell transfection, etc. In providing a kit. Another object of the present invention is to provide a method for separating DNA from agarose gel or polyacrylamide gel using the column kit.

The above object of the present invention is a chemically inert substance, resistant to weak alkaline weak alkali, excellent absorption of moisture from 1.0μM particles, excellent loading capacity, can be autoclavable, so there is little pollution problem even in long-term storage A glass microfiber column kit was prepared using borosilicate, a boron compound of silicate having a high purity, and plasmid DNA of high purity was prepared by using the excellent DNA adsorption capacity of the borosilicate glass microfiber without additional purification. The plasmid DNA was used to evaluate and analyze the superiority of the glass microfiber column kit of the present invention. Hereinafter, the configuration and operation of the present invention.

Figure 1a is an exploded view of the glass microfiber membrane multilayer kit (GF mini column kit).

FIG. 1B is an assembled sectional view of the column kit shown in FIG. 1A.

2A is an exploded view of a glass microfiber membrane / particle column kit (GP midi column kit).

FIG. 2B is an assembled sectional view of the column kit shown in FIG. 2A.

Figure 3 is a graph comparing the yield of plasmid DNA according to the material of the borosilicate glass microfibers and silicate glass microfibers.

Figure 4 is a graph showing the change in plasmid DNA yield with the increase of the glass microfiber membrane.

Figure 5 is a graph showing the change in plasmid DNA yield with increasing the glass microfiber particles.

Figure 6 is a restriction enzyme digestion of plasmid DNA prepared with a column kit of the present invention.

Figure 7 is a 35S dATP sequencing analysis of the plasmid DNA prepared by the column kit of the present invention.

Figure 8a is a photograph showing the staining reaction by β-galactosidase protein production during transfection of the plasmid DNA prepared with a conventional column kit.

Figure 8b is a photograph showing the staining reaction by β-galactosidase protein production during transfection of the plasmid DNA prepared by the column kit of the present invention.

9 is an analysis table of automatic sequencing analysis of pGEM 7Z plasmid DNA prepared by the column kit of the present invention using a T7 primer.

10 is an analysis table of the automatic sequencing analysis of pGEM 7Z plasmid DNA prepared by the kit of the present invention using Sp6 primer.

Figure 11 is a photograph of the DNA band from the agarose gel and separated using a GF mini column kit and electrophoresis on the agarose gel.

The present invention provides a method for preparing a glass microfiber membrane multilayer column and a glass microfiber membrane / particle column, preparing a plasmid DNA using the prepared column kit. The prepared plasmid DNA consists of restriction enzyme digestion, cell transfection and transduction, 35S sequencing analysis, and automatic sequencing analysis. Hereinafter, the specific configuration and operation of the present invention will be described with reference to Examples, but the scope of the present invention is not limited only to the following Examples.

Example 1

Preparation of glass microfiber column and obtaining plasmid DNA

Experimental Example 1. Preparation and use of glass microfiber membrane multilayer column for GF mini column kit

Borosilicate galss microfiber made of 8mm diameter circle, and several layers were stacked into 2-10mm plural layers (13) and inserted into a small spin mini column 11 (13mm in diameter and 31mm in length) and then 7.5mm ring Insert (12) to prepare the contents to fix and autoclave (Fig. 1a). When using the prepared glass fiber column is used in the collecting tube 10 (Fig. 1b). The multi-layer column of borosilicate glass microfiber membrane improves the purity and yield of plasmid DNA so that the thickness of the membrane increases rapidly from 2mm, so that the yield of plasmid DNA is up to about 200µg at 8mm as shown in FIG. Plasmid DNA of was obtained. On the other hand, the yield was not increased any more than 10mm.

Experimental Example 2. Fabrication and use of glass microfiber membrane / particle column for GP midi column kit

Borosilicate glass microfibers are made of glass microfiber particle suspension in a 10 mM Tris / HCl pH 7.5, 1 mM EDTA solution and a blender. 2 mm diameter 8 mm round glass microfiber particles 24 were placed in a 15 mm diameter, 80 mm long polypropylene medium column 22 shown in FIG. 2a, and then a glass microfiber particle suspension was added and spinned on a swing rotor centrifuge to obtain 5-10 mm. A glass microfiber membrane / particle dual column layer is made and the contents are fixed and autoclave with an 8 mm diameter ring or 8 mm frit (23) diameter. When using the prepared glass fiber column is used to support the Eppendorf tube 21 put into the collecting tube 20 (Fig. 2b). The plasmid DNA yield with increasing free microfiber membrane and the plasmid DNA yield with increasing capacity of free microfiber particles were increased as shown in FIGS. 3 and 4, respectively.

Experimental Example 3. Comparison of plasmid DNA yield by borosilicate glass microfiber column and silicate glass microfiber column

In order to compare the yield of plasmid DNA according to the material of borosilicate glass microfiber and silicate glass microfiber, a column was prepared in the same manner as in Experimental Example 1 and E. Plasmid DNA was prepared from the coli culture, and the yield and purity were analyzed. As a result, as shown in FIG. 5, the yield in the silicate glass microfiber column was reduced by about 20% compared to the borosilicate glass microfiber column, and the purity was also OD ₂₆₀ /. As OD ₂₈₀ = 1.5, the plasmid DNA purity OD ₂₆₀ / OD ₂₈₀ = 1.7-1.8 prepared by borosilicate glass microfiber column was not met.

Experimental Example 4. Comparison of yield and purity of plasmid DNA prepared by silica gel column and silica gel / borosilicate glass microfiber dual layer column

As in Example 2, five midi columns were filled with about 8 mm of silica gel, and the other five midi columns had a borosilicate glass microfiber membrane / particle layer of about 2 mm. After again making a silica gel layer of about 6mm layer and analyzed by preparing plasmid DNA as in Experimental Example 2 are shown in Table 1 below.

Purity and yield of plasmid DNA by column Type of column E.coli JM109 culture OD ₂₆₀ / OD ₂₈₀ (purity) Yield silica gel 200 ml 1.5-2.2 40 to 80 µg silica gel / borosilicate glass microfiber 200 ml 1.5-1.8 70 to 90 µg

As shown in Table 1, the yield and purity of the borosilicate glass microfiber layer in the silica gel column were significantly increased compared to the silica gel alone. Hereinafter, the plasmid DNA provided in all the experiments used what was manufactured by the borosilicate glass microfiber column.

Example 2

Plasmid DNA Preparation

Plasmid DNA was prepared using the GF mini column kit (or GP midi column kit) prepared in Example 1. E. coli strains containing plasmids were cultured and the strain culture was centrifuged to obtain pellets. This pellet was dissolved in P1 solution (50mM Tris, 10mM EDTA pH 8.0, 100µg / ml RNase A.), and then cell lysis in P2 solution (20mM NaOH, 1.0% SDS (W / V)) followed by P3 solution (3.2). mM Potasium acetate, pH 5.0). The neutralized solution was centrifuged to bind the supernatant to the DNA in the GF (or GP) column kit, followed by removal of nuclease, washing and eluting to obtain high purity plasmid DNA. At this time, to remove nuclease, 40% isopropanol, 4.2M guanidine hydrochloride was used and 10mM Tris / HCl (pH 7.5), 50mM NaCl, 0.1mM EDTA, 70% Ethanol was used as a washing solution.

Example 3

Molecular Biology Evaluation and Analysis of Prepared Plasmid DNA

Experimental Example 1.Restriction enzyme digestion

As a result of digesting the plasmid DNA prepared in Example 2 with restriction enzymes EcoRI and Hind III, effective DNA digestion was achieved as shown in FIG. 6.

Experimental Example 2. 35S sequencing analysis

10 μg of the plasmid DNA prepared in Example 2 and 10 μg of the plasmid DNA prepared in the conventional product were reacted as a Sequenase ^™ version 2.0 DNA sequencing kit (United State Biochemical.) Using α35S d-ATP, and then separated from the sequencing gel. autoradiography. As a result, as shown in Figure 7, the sequencing analysis of the DNA produced by the column kit of the present invention is clearly separated from the DNA band (Fig. 7A), while the sequencing analysis of the DNA prepared by the conventional column kit product (Qiagen) In the DNA band was not effectively separated (Fig. 7B).

Experimental Example 3. Cell transfection and transduction

Cell transfection and transduction were performed using the column kit of the present invention prepared in Example 1 and the conventional column kit (Qiagen). As a result, as shown in Figures 8a and 8b, when transfected plasmid DNA prepared with the column kit of the present invention effective protein expression in almost all cells were stained (Fig. 8b) while the conventional column kit When transfection of the DNA prepared with (Qiagen) was not effective protein expression (Fig. 8a).

Experimental Example 4. Automatic sequencing analysis

ABI PRIM ^TM Dye terminator cycle sequencing ready reaction kit (Perkin Elmer) was carried out using a 20 μl reaction volume of 8 μl terminal ready reaction mix, 0.4 μg template DNA and 3 pmole of primer at 96 ℃ / 30min, 50 ℃ / 15sec, The reaction was carried out at 60 ° C./4min for 25 cycles in Bio Rad Gene Cycler ^™ , precipitated as ethanol, dried in speed vac and analyzed in an automatic sequencer (Applied Biosystem). As a result, very effective sequence analysis was performed as shown in FIGS. 9 and 10. In this case, the template used above is a 1.2 kb cloned DNA containing pGEM7Z, and the primer is a T7 primer or an SP6 primer.

Experimental Example 5 DNA Separation from Agarose Gel or Polyacrylamide

DNA is isolated by electrophoresis from agarose gel / TAE or TBE, cut the band into a dialysis bag and filled with TAE solution, followed by electrophoresis to separate DNA from the gel. Take a solution, add the same volume of WA solution (40% isopropanol, 4.2M guanidine hydrochloride), add spin to the GF mini column of the present invention, and spin the WB solution (10 mM Tris / HCl pH 7.5, 30 mM EDTA, 70% ethanol). ), And then add 50 µl DW and spin to separate. Experimental results showed excellent DNA separation performance as shown in FIG.

Therefore, the efficiency of the column kit of the present invention manufactured through the above embodiments is as summarized in Table 2, the advantages are as follows. First, a simple and quick to produce plasmid DNA, and the second, DNA yield is extremely superior to GF mini column kit in to obtain a DNA amount of up to about 50 _to 100 times _(~ 200 ㎍) than the conventional spin column kit In the case of GP midi column kit, DNA amount of about 800 µg can be obtained. Third, purity of plasmid DNA prepared from glass microfiber of the present invention using GF mini column kit by measurement of spectophotometer (OD ₂₆₀ / OD ₂₈₀ ) = 1.7 _~ 1.8, GP midi kit using OD ₂₆₀ / OD ₂₈₀ = 1.6 _~ 1.7 high purity plasmid DNA can be obtained. Fourth, it can effectively block nuclease activity because it removes nucleases commonly found in E. coli and carbohydrates appearing in JM109 strain.

Efficiency of Prepared Plasmid DNA Manufacturing method E.coli JM109 culture Plasmid type OD ₂₆₀ / OD ₂₈₀ (purity) Maximum DNA Yield GF mini column kit 100-200ml pGem 7z 1.7-1.8 ~ 200 µg GP midi column kit 500 ml pGem 7z 1.6-1.7 ~ 800 µg

As described in detail in the above embodiment, the glass microfiber column of the present invention has the effect of obtaining a large amount of high purity plasmid DNA by simply and quickly preparing the plasmid, and the prepared plasmid DNA is excellent in purity and further purification. There is an effect that can be used in a variety of reactions without. In addition, the column kit of the present invention can be used to effectively separate DNA from agarose gel or polyamide gel, which is a very useful invention in the biopharmaceutical industry.

Claims (6)

Glass microfiber membrane column kit using glass microfiber membrane for the purpose of plasmid DNA production or glass microfiber particle column kit using glass microfiber particle. The glass microfiber membrane column kit of claim 1, wherein the glass microfiber membrane column kit comprises a minispin column tube, a collecting tube, a ring or frit, a glass microfiber membrane column, or the glass microfiber particle column kit comprises a midi column tube, a collecting tube, A column kit comprising a membrane consisting of an Eppendorf tube, a ring or frit, and a glass microfiber membrane / particle column. The column kit according to claim 1 or 2, wherein the glass microfiber membrane / particle column is made of borosilicate alone or a mixture of silicate and borosilicate. Method for preparing plasmid DNA using borosilicate glass microfiber membrane column or borosilicate glass microfiber particle column. DNA separation method from agarose or polyacrylamide gel using borosilicate glass microfiber membrane or borosilicate glass microfiber particle. A borosilicate glass microfiber membrane / particle layer in which borosilicate alone or a mixture of silicate is laminated on a lower portion of a silica gel column.